Project Summary Spinal interneurons integrate noxious and innocuous sensory inputs to the dorsal horn and gate their access to spinal projection neurons. Although specific cells within dorsal horn circuits have been identified and characterized, the diversity of interneuron subtypes, together with limited knowledge on their neurochemistry and connectivity, currently precludes comprehensive network mapping. Our long-term objective is to define changes in dorsal horn circuits under conditions of chronic pain. The objective of this exploratory project is to identify and comprehensively characterize interneurons that are presynaptic to nociceptive-specific (high- threshold, HT) and wide dynamic range (WDR) projection neurons. We will employ a novel approach that combines the following components: 1) Monosynaptic transfer of non-toxic modified rabies virus (SiR) will be used to induce expression of fluorescent reporter genes and calcium indicators in synaptically connected neurons. 2) Calcium imaging in spinal cord slices with attached dorsal roots (DR) will be used to monitor responses of identified dorsal horn neurons to defined primary afferent input. 3) Dual patch-clamp recording from identified synaptically connected neurons will be integrated with Patch-seq methodology for single-cell transcriptome analysis. Information regarding the complement of inhibitory and excitatory neurons synapsing on dorsal horn projection neurons is limited. Further, data on differences in this complement based on the functionality (HT, WDR) of projection neurons is lacking. Such knowledge would expand our understanding of excitatory/inhibitory balance in modality-specific dorsal horn circuits converging on projection neurons and increase the possiblilty of circuit-specific neuromodulation for the treatment of chronic pain. The central hypothesis of this proposal is that HT and WDR projection neurons receive synaptic input from distinct subpopulations of interneurons. In Aim 1, we will define subpopulations of interneurons that synapse on projection neurons using monosynaptic SiR transfer, imaging of calcium responses to dorsal root stimulation in spinal cord slices, and CLARITY-based neuroanatomical analysis. In Aim 2, we will test the hypothesis that subpopulations of interneurons synapsing on HT and WDR projection neurons can be distinguished by transcriptomic analysis. Following delivery of SiR to projection neurons and its monosynaptic transfer to their presynaptic partners, excitatory and inhibitory connections between synaptic pairs will be characterized using patch-clamp electrophysiology in spinal cord slices and patch-seq methodology will be employed for single-cell RNAseq transcriptomic analysis of recorded neurons. This project will generate critical new data on the connectivity of spinal projection neurons to the network of interneurons that process primary afferent inputs and will lay the groundwork for comprehensive anatomical and functional mapping of dorsal horn circuits and their plasticity under chronic pain conditions.